DESCRIPTION: This research project describes the investigation of oxygen activation by nonheme diiron enzymes. Four metalloenzymes are selected for study: (i) protein R2 of ribonucleotide reductase, (ii) the hydroxylase component of methane monooxygenase, (iii) stearoyl-acyl carrier protein delta9-desaturase, and (iv) the fast ferroxidase site in ferritin. These enzymes are evolutionarily related, possess structurally similar metal sites, and activate oxygen, but they catalyze diverse oxidation reactions. Crystal structures exist for the reduced and/or resting native enzymes and some mutants, but only sparse structural information is available on reaction intermediates. A hypothesis to be explored is that the iron centers adopt a common reaction pathway, namely, that the diferrous enzyme binds oxygen to form an initial peroxodiferric species, P, and 0-0 bond cleavage leads to a high-valent diiron(IV) intermediate. In MMOH, the diiron(IV) species, Q, is the active catalyst and is proposed to have a diamond-core structure. In protein R2, one-electron reduction leads to mixed valence [F(II)F(III)] intermediate, X, as the catalytically competent species. In ferritin, the peroxodiferric species may lose hydrogen peroxide to leave an oxidized metal cluster for mineralization. We describe the use of resonance Raman, optical absorption, EPR, and Mossbauer spectroscopy to investigate the structures of intermediates P, Q, and X. Enzyme intermediates can be trapped by rapid freeze-quench methods for spectroscopic analysis. Vibrational modes of iron-oxo and radical species known or suspected to occur during these reactions can be identified. Oxygen isotopes will be used to assign vibrational modes from mass-dependent shifts and to provide details on chemical bonding and binding geometry. The present proposal builds on the first successful identification of a common mu-1,2-peroxo intermediate in wild-type ferritin freeze-trapped at 25 milliseconds, as well as in a mutant of R2, and chemically reduced delta9-desaturase. An overall aim is to understand how enzymes with similar active sites carry out diverse reactions. This research will expand our knowledge of oxygen activation as well as mechanisms of oxygen and free-radical toxicity.